PROJECT SUMMARY/ABSTRACT Mycobacterium tuberculosis (Mtb) requires the ESX-5 specialized protein secretion system for both nutrient uptake and virulence, but the ESX-5 effectors that directly influence nutrient acquisition or interactions with host cells are currently undefined. The long-term goal is to understand the mechanisms that enable Mtb to evade host immune defenses to establish and maintain persistent infections. The objective of this proposal is to identify ESX-5 substrates that impact Mtb pathogenesis. The proposed research will test the central hypothesis that ESX-5 secretes many substrates and that proteins encoded within the esx-5 locus directly influence Mtb virulence, independent of a function in nutrient acquisition. This proposal will utilize unique genetic tools to dissect the function of the ESX-5 secretion system. Preliminary data demonstrate substantial alterations in the secreted protein profile of a mutant that hyper-secretes the known ESX-5 substrates. Specific Aim 1 will make use of this ESX-5 hyper-secretion mutant, growth conditions that stimulate ESX-5 secretion and strains that conditionally express core components of the ESX-5 secretion system to globally identify ESX- 5 substrates by a proteomics approach. Specific Aim 2 will test the importance of the mycobacteria-specific PE, PPE and Esx proteins that are encoded within the esx-5 locus for Mtb virulence using a series of Mtb deletion mutants and both mouse and macrophage infection models. The results of these studies will significantly enhance our understanding of the Mtb host-pathogen interaction by revealing proteins that are secreted via ESX-5 and identifying individual ESX-5-associated proteins that are required for virulence. Identification of functional ESX-5 effectors will enable detailed molecular characterization of the mechanisms by which these proteins manipulate host cell functions to facilitate design of novel antimicrobials to combat tuberculosis infection.